Physicochemically and pharmacokinetically stable nonapeptide kiss1 receptor agonists with highly potent testosterone-suppressive activity

Article abstract of DOI:10.1021/jm5005489

Modifications of metastin(45-54) produced peptide analogues with higher metabolic stability than metastin(45-54). N-terminally truncated nonapeptide 4 ([d-Tyr⁴⁶,d-Pya(4)⁴⁷,azaGly⁵¹,Arg(Me) ⁵³]metastin(46-54)) is a representative compound with both potent agonistic activity and metabolic stability. Although 4 had more potent testosterone-suppressant activity than metastin, it possessed physicochemical instability at pH 7 and insufficient in vivo activity. Instability at pH 7 was dependent upon Asn⁴⁸ and Ser⁴⁹; substitution of Ser ⁴⁹ with Thr⁴⁹ reduced this instability and maintained KISS1 receptor agonistic activity. Furthermore, [d-Tyr⁴⁶,d-Trp ⁴⁷,Thr⁴⁹,azaGly⁵¹,Arg(Me)⁵³,Trp ⁵⁴]metastin(46-54) (14) showed 2-fold greater [Ca²⁺] _i-mobilizing activity than metastin(45-54) and an apparent increase in physicochemical stability. N-terminal acetylation of 14 resulted in the most potent analogue, 22 (Ac-[d-Tyr⁴⁶,d-Trp⁴⁷,Thr ⁴⁹,azaGly⁵¹,Arg(Me)⁵³,Trp⁵⁴] metastin(46-54)). With continuous administration, 22 possessed 10-50-fold more potent testosterone-suppressive activity in rats than 4. These results suggested that a controlled release of short-length KISS1 receptor agonists can suppress the hypothalamic-pituitary-gonadal axis and reduce testosterone levels. Compound 22 was selected for further preclinical evaluation for hormone-dependent diseases.

Full text of DOI:10.1021/jm5005489

Article
pubs.acs.org/jmc
Physicochemically and Pharmacokinetically Stable Nonapeptide
KISS1 Receptor Agonists with Highly Potent Testosterone-
Suppressive Activity
Taiji Asami,*,† Naoki Nishizawa,† Hisanori Matsui,† Yoshihiro Takatsu,† Atsuko Suzuki,† Atsushi Kiba,†
Michiko Terada,† Kimiko Nishibori,† Masaharu Nakayama,† Junko Ban,‡ Shin-ichi Matsumoto,†
Naoki Tarui,† Yukihiro Ikeda,† Masashi Yamaguchi,† Masami Kusaka,‡ Tetsuya Ohtaki,†
and Chieko Kitada†
†Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., Fujisawa, Kanagawa 251-8555, Japan
‡Pharmaceutical Production Division, Takeda Pharmaceutical Company Ltd., Osaka 532-8686, Japan
S
* Supporting Information
ABSTRACT: Modifications of metastin(45−54) produced peptide analogues with higher
metabolic stability than metastin(45−54). N-terminally truncated nonapeptide 4 ([D-
Tyr46,D-Pya(4)47,azaGly51,Arg(Me)53]metastin(46−54)) is a representative compound
with both potent agonistic activity and metabolic stability. Although 4 had more potent
testosterone-suppressant activity than metastin, it possessed physicochemical instability at
pH 7 and insufficient in vivo activity. Instability at pH 7 was dependent upon Asn48 and
Ser49; substitution of Ser49 with Thr49 reduced this instability and maintained KISS1
receptor agonistic activity. Furthermore, [D-Tyr46,D-Trp47,Thr49,azaGly51,Arg-
(Me)53,Trp54]metastin(46−54) (14) showed 2-fold greater [Ca2+]i-mobilizing activity
than metastin(45−54) and an apparent increase in physicochemical stability. N-terminal
acetylation of 14 resulted in the most potent analogue, 22 (Ac-[D-Tyr46,D-
Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46−54)). With continuous administra-
tion, 22 possessed 10−50-fold more potent testosterone-suppressive activity in rats than
4. These results suggested that a controlled release of short-length KISS1 receptor
agonists can suppress the hypothalamic−pituitary−gonadal axis and reduce testosterone levels. Compound 22 was selected for
further preclinical evaluation for hormone-dependent diseases.
In preclinical studies, single administration of metastin
markedly stimulated FSH/LH release in rat models,13 and
continuous administration reduced testosterone levels.14 The
molecular mechanisms for KISS1R signaling suggest that
appropriate dosing of KISS1R agonists should activate or
suppress the HPG axis, thereby potentially preventing or
treating a number of sex-hormone-dependent diseases.
An N-terminally truncated metastin(45−54) analogue has
demonstrated biological activity in vitro.1 This fragment
peptide was 3−10 times more potent than metastin, whereas
the truncated metastin(46−54) was less potent than full-length
metastin,5 indicating that the core residues for biological
activity are located in C-terminal short peptides.
INTRODUCTION
■
Metastin/kisspeptin, a 54-amino acid peptide, is a ligand of the
orphan G-protein-coupled receptor hOT7T175, also known as
AXOR12, and GPR54, currently renamed the KISS1 receptor,
KISS1R.1−6 KISS1R is most closely related to the galanin
receptor family with approximately 45% homology; however, it
binds neither galanin nor galanin-like peptide.7 Several
neuropeptides, including the RFamide and RWamide families,
have been identified as ligands for KISS1R.8 Metastin neurons
have been mapped in several species, including fish, rodents,
and humans. The strongest KISS1R expression has been
observed in hypothalamic tissues, with little expression in other
brain areas.4−11
In our previous studies, rational modification of meta-
stin(45−54) resulted in [D-Tyr45,Arg(Me)53]metastin(45−54)
(2), which demonstrated higher metabolic stability than
metastin(45−54).15,16 Substitutions of D-Tyr at position 45
and Nω-methylarginine [Arg(Me)] at position 53 eliminated N-
terminal and C-terminal degradation, respectively, and retained
Although first described in the context of cancer meta-
stasis,1,2 metastin−KISS1R signaling has subsequently been
shown to play a vital role in reproduction via regulation of the
HPG axis. In 2003, two independent studies reported that
inactivation mutations of KISS1R led to idiopathic hypogona-
dotropic hypogonadism in humans and mice.9,10 Further
research suggests that metastin potently elicits GnRH and
gonadotropin secretion in mammals and non-mammalian
species.4,12
Received: April 8, 2014
Published: June 11, 2014
© 2014 American Chemical Society
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KISS1R activity. Metabolic stability was further improved by
azaGly51 substitution; deletion of Asn46 and substitution of
Trp47 produced highly potent and long-acting KISS1R agonists,
e.g., [D-Tyr46,D-Pya(4)47,azaGly51,Arg(Me)53]metastin(46−54),
compound 4.17
preparations. Intramolecular formation of an Asu peptide
intermediate is known to be the first step in these reactions, as
shown in Figure 1. The mechanism of succinimide formation
Compound 4 was the first short-length metastin analogue to
show testosterone suppression activity in vivo, as evidenced by
reduced plasma testosterone levels in male rats upon
continuous administration of 4.17 This result suggested that
metabolically stable metastin analogues could form the basis for
the development of therapeutic candidates for hormone-
dependent diseases such as prostate cancer.
The chemical stability of 4, containing Asn−Ser residues, was
investigated using a range of analytical techniques.18 Hydrolysis
was monitored by changes in mass, size, charge, hydro-
phobicity, and UV absorption. Compound 4 was incubated at
37 °C in aqueous buffer solutions at four different pH levels.
Analyses by reversed-phase HPLC showed the deamidation of
Asn48 of 4 with the formation of the two aspartyl derivatives [D-
Tyr46,D-Pya(4)47,Asp48,azaGly51,Arg(Me)53]metastin(46−54)
and [D-Tyr46,D-Pya(4)47,β-Asp48,azaGly51,Arg(Me)53]metastin-
(46−54). Residual compound 4 was quantified by UV
absorption via HPLC chromatography. 4 was stable in buffer
solutions of pH 3 and 5; however, deamidation was observed at
pH 1.2 and 7, and the rate of deamination was temperature-
dependent. The decomposition products obtained at pH 7 were
assumed to be Asp48 and β-Asp48 derivatives by LC−MS
molecular weight and HPLC retention time compared with
those of known compounds.19,20 These results suggested that
physicochemically more stable analogues would be needed for
the development of useful therapeutic candidates.
Figure 1. Scheme of the deamidation reaction of Asn-containing
metastin analogue 4 in aqueous solution.
involves deprotonation of the backbone amide of Ser49 followed
by attack of the anionic nitrogen on the carbonyl group of
Asn48. Neighboring amino acid residues that allow for chain
flexibility and hydrogen-bonding interactions, such as Gly or
Ser, facilitate the formation of the Asu intermediate. The
subsequent hydrolysis of the Asu intermediate occurs on either
side of the imide nitrogen, leading to the formation of α-
aspartyl (Asp) or β-aspartyl (or isoaspartic acid, isoAsp). The
rate of this degradation process is dependent upon the primary
sequence of the peptide, temperature, buffer pH, and
concentration.23−28
To initiate this research, we synthesized compound 5 where
Phe54 of compound 4 was substituted with Trp54. In our
previous metastin research, Trp54 analogue 1 had shown the
highest receptor affinity (data not shown). The substitution of
Phe54 with Trp54 resulted in 3-fold greater in vitro human
activity of 5 compared with 4, as shown in Table 1. On the
other hand, in vitro rat activities of 4 and 5 were comparable
(Table 1). However, plasma testosterone concentrations were
below the lower limit of detection (0.04 ng/mL) in two of five
rats when 5 was administered at a dose of 0.3 nmol/h (zero of
five rats after dosing of 4) (Table 2). It is supposed that the
higher in vivo activity of 5 than 4 was dependent on its
In this study, we designed and synthesized metastin
analogues that show greater agonistic activities and phys-
icochemically and pharmacokinetically more stable profiles than
those of compound 4.
RESULTS
■
Chemistry. All peptides were synthesized by standard
Fmoc-based solid-phase synthetic methods. The obtained crude
peptides were purified to homogeneity using preparative
HPLC. The purity of each peptide was ascertained by analytical
HPLC, and the structure assignment was performed by
MALDI-TOF MS.
Biological Activity. Synthesized analogues were charac-
terized in two assays of human KISS1R (hKISS1R) receptor
activity using an intracellular calcium mobilization FLIPR assay
and a cell membrane binding assay. The FLIPR assay used
CHO cells stably expressing recombinant hKISS1R, and the
binding assay used membrane fractions isolated from hKISS1R-
expressing CHO cells.
The maximum response observed in the presence of
compound was corrected for baseline measurements and
expressed as a percentage of the corrected response to a
maximal concentration of control metastin(45−54). Nonlinear
regression of normalized data was performed to estimate the
potency (EC50) of selected compounds.
Design and Synthesis of Nonapeptide Metastin
Analogues Substituted at Positions 48 and 49. The
irreversible, spontaneous, and nonenzymatic deamidation of
Asn residues is one of the major chemical degradation pathways
for peptides and proteins. This occurs frequently during
synthesis, purification, sequencing, and storage21,22 and
introduces undesired heterogeneity into peptide and protein
pharmacokinetic profiles, such as CLtotal
.
To reduce deamination, further chemical modifications of 5
comprising substitutions of Ser49 with β-branched amino acids
such as Thr (6), Val (7), Phg (8), and Tle (9) were carried out.
Trp49 analogue 10 was also synthesized as a representative
compound with a bulky side chain. Substitution at position 49
with cyclic amino acids such as Pro (11) and trans-4-Hyp (12)
was expected to avoid cyclic imide formation completely.
All amino acid replacements at position 49 of 5 showed
excellent [Ca2+]i-mobilizing activities for hKISS1R (Table 1).
Among compounds with β-branches or a cyclic amino acid at
position 49, the Thr (6), Val (7), and Pro (11) derivatives had
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Table 2. Testosterone-Suppressive Activities of Nonapeptide Metastin Analogues H-D-Tyr-AA47-AA48-AA49-Phe-azaGly-Leu-
Arg(Me)-AA54-NH2 Substituted between Positions 47 and 54
a
testosterone-suppressive activity, rats with testosterone levels of <0.04 ng/mL
[n (%)]
compd
AA47
AA48
AA49
AA54
2 nmol/h
0.5 nmol/h
0.3 nmol/h
0.1 nmol/h
4
5
D-Pya(4)
D-Pya(4)
D-Pya(4)
D-Pya(4)
D-Trp
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Ala
Ser
Ser
Val
Pro
Ser
Thr
Val
Ser
Ser
Phe
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
5 (100)
4 (80)
4 (80)
3 (60)
3 (60)
0
0
0
0
2 (40)
b
b
7
ND
ND
b
b
b
11
13
14
15
16
20
ND
1 (20)
4 (80)
3 (60)
0
ND
ND
b
ND
2 (40)
3 (60)
0
0
D-Trp
5 (100)
b
b
b
D-Trp
ND
ND
ND
b
b
b
D-Pya(4)
D-Trp
0
0
ND
ND
ND
b
b
b
Abu
ND
ND
ND
a
Plasma testosterone-suppressive activities of peptide analogues at 2, 0.5, 0.3, or 0.1 nmol/h. Peptides were dissolved in distilled water to prepare 2,
0.5, 0.3, or 0.1 mM solutions used in ALZET osmotic pumps implanted subcutaneously in five Crl:CD(SD) male rats. After 6 days, plasma
testosterone levels were measured using RIA. Testosterone suppression was expressed as the number of rats with plasma testosterone levels below
b
the limit of detection (0.04 ng/mL). Not determined.
better agonistic activities in the FLIPR hKISS1R assay than the
other peptides. The in vivo activity of selected compounds was
evaluated by testosterone-suppressive activity in rats (Table 2).
Six days of continuous administration of compound 7 at 2
nmol/h attenuated the plasma testosterone concentration in
rats; however, at 0.5 nmol/h, 7 demonstrated less activity than
5. Three D-Trp47 analogues (13, 14, 15) with Ser49, Thr49, and
Val49, respectively, were expected to improve KISS1R agonistic
activities due to the hydrophobic interaction at the side chain of
D-Trp47 with KISS1R. All derivatives exhibited high functional
activities of both human and rat receptors (Table 1). [D-
Trp47,Thr49] analogue 14 possessed higher agonistic activity
than 5. Moreover, 14 reduced plasma testosterone at a dose of
0.3 nmol/h. On the other hand, Val49 derivative 15 dosed at 0.5
nmol/h did not reduce the testosterone concentration in rats.
Since the Asn48 residue caused the decomposition of 4, the
Ala (16) and Thr (17) analogues of 5 were synthesized to
avoid the deamidation reaction. Ala analogue 16 retained
agonistic activity similar to that of 5 for both the human and rat
receptor. However, 2 nmol/h continuous administration of 16
was not associated with reduced testosterone concentration in
rats. Combined amino acid replacements at positions 47 and 48
yielded three analogues, [D-Trp47,Ala48,Ser49] (18), [D-
Trp47,Ala48,Thr49] (19), and [D-Trp47,Abu48,Ser49] (20),
which showed excellent agonistic activity. However, 2 nmol/h
continuous administration of 20, which possessed the highest in
vitro activity of these three analogues in rats, did not suppress
plasma testosterone in vivo. On the basis of the results of
compounds 16 and 20, the Asn48 residue was proposed to be
essential for testosterone-suppressive activity in vivo.
(Table 3). The deamination rate of 13 at pH 7 was as fast as
that of 4, with 28.0% of the product remaining after a 2-week
Table 3. Stability Studies of Ser49 Analogues 4 and 13, and
Thr49 Analogue 14 in Aqueous Solution
a
residual ratio (%)
compd
time
pH 1.2 (JP1)
pH 3
pH 5
pH 7
4
initial
100.0
60.3
32.8
100.0
60.8
33.4
100.0
64.9
39.1
100.0
99.4
100.0
94.8
91.2
100.0
96.0
90.8
100.0
98.8
96.5
100.0
54.9
28.0
100.0
56.5
28.0
100.0
84.9
72.8
1 week
2 week
initial
100.5
100.0
98.2
13
14
1 week
2 week
initial
97.1
100.0
98.7
1 week
2 week
98.2
a
Aqueous solutions of peptides at pH 1.2 (JP1), 3, 5, or 7 were
incubated at 37 °C for up to 2 weeks. The amount of peptide in the
buffer solutions was determined from the area under the HPLC peaks.
The percentage of a given compound recovered from the buffer
solutions after incubation (stability index) was calculated relative to
the amount of peptide at time 0 taken as 100%.
incubation period. In contrast, 72.8% of 14 remained after a 2-
week incubation at pH 7. Thr49 replacement was therefore
considered to contribute to decreased succinimide formation
and improved solution stability in neutral buffer.
Design and Synthesis of Nonapeptide Metastin
Analogues Modified at the N-Terminus. Peptide metabo-
lism and excretion are associated with α-amino acid residues
with a free amino group at the N-terminus. As such, N-terminal
modifications were planned in an attempt to further improve
the testosterone-suppressive activity of 14. For N-terminal
modification, deaminated analogue 21 and acylated analogues
22−25 of 14 were synthesized. Acetylated 22, butylated 23,
cyclopropylcarbonylated 24, and benzoylated analogue 25 were
selected as compounds with representative acyl moieties, i.e.,
aliphatic, alicyclic, and aromatic moieties. The in vivo activities
of these peptides were evaluated using 50% DMSO as the
primary solvent due to the low water solubility of several of
these peptides.
We concluded that the combination of D-Trp47 and Thr49 in
14 gave an optimal core structure in terms of both stability in
solution and biological activity.
Physicochemical Properties of Metastin Analogues 13
and 14. The effect of Thr49 substitution on the deamination of
metastin analogues was evaluated by comparing the stability of
Ser49 analogue 13 in solution with that of Thr49 analogue 14.
Compounds 13 and 14 were incubated at 37 °C in aqueous
solution at four different pH values, 1.2, 3, 5, and 7.
The residual amount of 13 and 14 was quantified respectively
by UV absorption using HPLC chromatography. Although 13
and 14 were stable in buffer solution at pH 3 and 5,
deamination of 13 was observed in solution at pH 1.2 and 7
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All analogues possessed excellent agonistic activities, and the
[Ca2+]i-mobilizing activities were more potent than that of
metastin(45−54) (Table 4). Among these analogues, N-
terminal acetylated analogue 22 showed a significant increase
in testosterone-suppressive activities (Table 5). Plasma
testosterone concentrations in rats given 0.05 nmol/h of 22
were below the limit of detection (0.04 ng/mL) in RIA.
Moreover, the 0.03 nmol/h dose of 22 attenuated the
testosterone levels, with more than 20-fold higher activity
than 4. This improvement was thought to result from N-
terminal acetylation. The pharmacokinetic profiles of 22 are
discussed below.
Although other N-terminal acylated analogues, 23−25,
retained high in vitro activities, testosterone-suppressive
activities were weaker compared with that of the acetylated
analogue 22. Increased hydrophobicity of N-terminal acylated
analogues 23−25 may result in their lower BA after
subcutaneous administration. In addition, the in vivo activity
of N-terminal deaminated analogue 21 was less than that of 22,
although 21 had more potent agonistic activity than 22.
N-terminal acetylation was considered the most effective
approach for improving in vivo activity in this series of
peptides.29 The N-terminal acetamido moiety of 22 was
thought to contribute to not only the avoidance of N-terminal
cleavage but also the improved penetration into blood. The N-
terminal acetylated Pya(4)47 (26) and Phe54 (27) analogues
also attenuated the plasma testosterone concentration in all rats
tested at the 0.05 nmol/h dose.
Physicochemical Profiles of Metastin Analogues 22,
26, and 27. The physicochemical stability of the highly potent
analogues 22, 26, and 27 was evaluated using the same method
as for 13 and 14 described above. All peptides maintained
>95% purity after 2 weeks in pH 3 and 5 buffer solutions
(Table 6). The deamination rates of 22, 26, and 27 at pH 7
were lower than that of 4, as shown in Table 6, with 66.4%,
69.8%, and 67.6%, respectively, of product remaining after 2-
week incubations. Thr49 replacement contributed to decreased
succinimide formation and improved solution stability in
neutral buffer solution.
Pharmacokinetic Profiles of Metastin Analogues 14,
22, 26, and 27. The pharmacokinetic profiles of the prototype
key compound 14 and potent analogues 22, 26, and 27 were
evaluated using Crl:CD(SD) male rats. These peptides were
administered intravenously (Table 7) or subcutaneously (Table
8) at a dose of 1 mg/kg. Although the plasma stability of 4 was
high in vitro, 2 nmol/h continuous administration of 4 in
Copenhagen rats was very low (1.1 0.3 ng/mL).30 The renal
excretion of compound 4 via the urine was likely due to the
hydrophilic nature of 4. On the other hand, the value of VdSS of
22 was 3-fold lower than that of 14. 22 showed better
phamacokinetic parameters compared with 14 after intravenous
administration. The N-terminal acetylation effectively improved
the AUC values when administered subcutaneously. The
hydrophilic substitution of D-4-pyridylalanine [D-Pya(4)]47
(26) with D-Trp47 (22) resulted in a small improvement in
the BA value in subcutaneous administration. Another amino
acid replacement with a smaller side chain, Phe54 (27),
produced higher BA values than 22, although this replacement
was not useful in terms of VdSS and CLtotal values.
Continuous Administration of Metastin Analogues
22, 26, and 27 in Male Rats. Three selected analogues, 22,
26, and 27, were tested in an intact male rat assay (Figure 2).
The purpose of this assay was to examine the steady-state
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Table 5. Testosterone-Suppressive Activities of Nonapeptide Metastin Analogues R-AA46-AA47-Asn-Thr-Phe-azaGly-Leu-
Arg(Me)-AA54-NH2 Substituted at the N-Terminus, Positions 46, 47, and 54
a
testosterone-suppressive activity, number of rats with testosterone
levels of <0.04 ng/mL (max 5)
compd
R
AA46
D-Tyr
AA47
AA54
0.1 nmol/h
0.05 mol/h
0.03 mol/h
0.01 mol/h
b
b
b
4
14
21
22
23
24
25
26
27
D-Pya(4)
D-Trp
Phe
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Phe
0
0
5
ND
ND
ND
b
b
b
D-Tyr
ND
ND
ND
b
b
deaminoTyr
D-Tyr
D-Trp
ND
ND
0
0
b
Ac
D-Trp
ND
5
4
0
b
b
butyryl
D-Tyr
D-Trp
3
1
0
ND
ND
b
b
b
cyclopropylcarbonyl
D-Tyr
D-Trp
ND
ND
ND
b
b
b
benzoyl
Ac
D-Tyr
D-Trp
ND
ND
ND
b
b
D-Tyr
D-Pya(4)
D-Trp
ND
5
5
ND
2
0
b
Ac
D-Tyr
ND
4
a
Plasma testosterone-suppressive activities of peptide analogues at 2, 0.5, 0.3, or 0.1 nmol/h. Peptides were dissolved in 50% DMSO to prepare 2,
0.5, 0.3, or 0.1 mM solutions used in ALZET osmotic pumps implanted subcutaneously in five Crl:CD(SD) male rats. After 6 days, plasma
testosterone levels were measured using RIA. Testosterone suppression was expressed as the number of rats with plasma testosterone levels below
b
the limit of detection (0.04 ng/mL). Not determined.
Continuous administration of 26 and 27 at 30 or 100 pmol/h
for 1 week suppressed testosterone in all five rats. Four weeks
later, one of five rats dosed with 30 nmol/h of 26 or 100 nmol/
h of 27 did not decrease plasma testosterone to undetectable
levels. In contrast, all rats had decreased plasma testosterone to
undetectable levels when 22 was administered at 30 and 100
pmol/h. These results suggested that 22 was the most potent of
the three peptides. The minimum effective dose of 22 was 30
pmol/h in this study.
Single Administration of Compound 22 in Male Rats.
The effect of single subcutaneous administration of 22,
metastin, or metastin(45−54) on plasma LH in male rats was
examined at various concentrations from 0.01 to 1000 nmol/kg
(Figure 3). Comparing AUC data for 22 and metastin, 22 was
approximately 100-fold more potent than metastin in
stimulating the release of LH.
Plasma testosterone levels were also elevated following single
subcutaneous administration of 22, metastin, or metastin(45−
54) (Figure 4). At 0.01 nmol/kg 22 and 1 nmol/kg metastin or
metastin(45−54), plasma testosterone levels were sensitive to
changes in plasma LH levels. Thus, 22 had a potent effect on
LH and testosterone release in male rats, most likely due to its
improved agonistic activity against KISS1R and physicochem-
ical stability in vivo. Moreover, there were no significant issues
in preclinical toxicity studies in rats and dogs (data not shown).
Safety margins based on the 4-week continuous dosing studies
in rats and dogs were more than 1000-fold.
Table 6. Stability of Metastin Analogues 22, 26, and 27 in
Aqueous Solutions
a
residual ratio (%)
compd
time
pH 1.2 (JP1)
pH 3
pH 5
pH 7
22
initial
100.0
55.6
28.8
100.0
58.3
33.0
100.0
50.6
22.3
100.0
97.7
95.6
100.0
98.7
96.8
100.0
97.5
95.0
100.0
95.9
93.6
100.0
98.5
97.0
100.0
98.1
96.3
100.0
79.7
66.4
100.0
82.6
69.8
100.0
84.5
67.6
1 week
2 week
initial
26
27
1 week
2 week
initial
1 week
2 week
a
Aqueous solutions of peptides at pH 1.2 (JP1), 3, 5, or 7 were
incubated at 37 °C for up to 2 weeks. The amount of peptide in the
buffer solutions was determined from the area under the HPLC peaks.
The percentage of a given compound recovered from the buffer
solutions after incubation (stability index) was calculated relative to
the amount of peptide at time 0 taken as 100%.
concentration of plasma testosterone after 1 and 4 weeks of
continuous administration of metastin analogues. These results
provide a direct measure of the in vivo ability to suppress
testosterone in an intact animal model. All infused analogues
produced undetectable steady-state concentrations of testoster-
one for up to 1 week. Values below the limit of detection (0.04
ng/mL) in RIA were treated as 0.04.
a
Table 7. Pharmacokinetic Parameters of Metastin Analogues after Intravenous Administration in Rats
compd
4
14
22
26
27
C5min (ng/mL)
AUC0−24h [(ng·h)/mL]
MRT (h)
969 88
1443 86
503 39
2509 287
1225 79
2072 142
1262 73
0.69 0.1
547 37
794 45
1449 171
497 98
239
9
0.38
0
0.73 0.1
1455 227
1997 157
0.69
0
0.44 0.1
896 33
Vdss (mL/kg)
1577 110
4196 164
562 37
819 52
CLtotal (mL/h/kg)
2067 423
a
Peptides were administered intravenously to SD rats at a dose of 1 mg/kg. Blood samples were collected at 5, 10, 15, and 30 min and 1, 2, 4, 8, and
24 h after injection. Plasma was collected by removing plasma proteins using 0.2% formic acid/methanol (1:4, v/v) and acetonitrile, followed by
centrifugation at 15 000 rpm for 5 min. The obtained supernatant was treated with 0.4% aqueous formic acid and centrifuged at 15 000 rpm for 5
min. The plasma concentration of peptides was determined by LC−MS. C5min, AUC0−24h, MRT, Vdss, and CLtotal were obtained for each rat after
intravenous administration and are expressed as the mean standard deviation (n = 3).
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a
Table 8. Pharmacokinetic Parameters of Metastin Analogues after Subcutaneous Administration in Rats
compd
4
14
22
26
27
Cmax (ng/mL)
Tmax (h)
145 20
0.14 0.1
96 11
47
5
100 44
0.19 0.10
115 44
0.86 0.2
9.4 3.6
90 19
114 13
0.42 0.1
144 41
0.88 0.2
28.9 10.1
0.19 0.05
46
0.42 0.14
152 36
1.07 0.1
12.1 3.0
AUC0−24h [(ng·h)/mL]
MRT (h)
9
0.61 0.1
40.4 4.9
0.88 0.1
9.2 2.0
BA (%)
a
Peptides were administered subcutaneously to SD rats at a dose of 1 mg/kg. Blood samples were collected at 5, 10, 15, and 30 min and 1, 2, 4, 8,
and 24 h after injection. Plasma was collected by removing plasma proteins using 0.2% formic acid/methanol (1:4, v/v) and acetonitrile, followed by
centrifugation at 15 000 rpm for 5 min. The obtained supernatant was treated with 0.4% aqueous formic acid and centrifuged at 15 000 rpm for 5
min. The plasma concentration of peptides was determined by LC−MS. Cmax, Tmax, AUC0−24h, MRT, and BA were obtained for each rat after
subcutaneous administration and are expressed as the mean standard deviation (n = 3).
Figure 3. Effects of single subcutaneous administration of 22,
metastin, or metastin(45−54) on plasma LH in male rats at 0, 0.01,
0.1, 1, 10, or 1000 nmol/kg. Compound 22, metastin, or metastin(45−
54) was dissolved in 50% DMSO and administered subcutaneously in
five Crl:CD(SD) male rats. LH AUC0−4h was measured using blood
samples (0.4 mL) obtained from the tail vein of 22-, metastin-, or
metastin(45−54)-treated animals before administration (0 h) and 0.5,
1, 2, 4, and 8 h after administration. Data are expressed as the mean
standard deviation (n = 6). A pound sign and two asterisks indicate p
≤ 0.025 (one-tailed Shirley−Williams test) and p ≤ 0.01 (Aspin−
Welch t test) vs vehicle (0 nmol/kg) group, respectively.
Figure 2. Plasma testosterone-suppressive activities by continuous
administration of compound 22, 26, or 27 in male rats at 0, 10, 30, or
100 pmol/h. Compound 22, 26, or 27 dissolved in 50% DMSO was
diluted in distilled water to prepare 10, 30, or 100 μM peptide
solutions added to ALZET osmotic pumps which were implanted
subcutaneously in five Crl:CD(SD) male rats. After 1 or 4 weeks, the
plasma testosterone level of each rat was measured using RIA. The
value below the limit of detection (0.04 ng/mL) in RIA was treated as
0.04. Each bar represents the mean standard deviation of plasma
testosterone from five rats. A pound sign indicates p ≤ 0.025 (one-
tailed Shirley−Williams test) vs vehicle (0 pmol/h) group.
Figure 4. Effects of single subcutaneous administration of 22,
metastin, or metastin(45−54) on plasma LH in male rats at 0, 0.01,
CONCLUSION
■
This study of metastin(45−54) analogues, designed to improve
their pharmacokinetic and physicochemical properties, led to
the identification of a novel reduced-sized metastin analogue,
22. Compound 22 was 10−50-fold more potent than 4 in vivo.
Three key replacements of N-terminal acetyl, D-Trp47, and
Trp54 in 22 were conducive to the production of a long-acting
analogue with low CLtotal. These replacements significantly
contributed to both improved metabolic stability and inherent
potency in increasing testosterone suppression in vivo.
Continued administration of KISS1R agonists suppressed
0.1, 1, 10, or 1000 nmol/kg. Compound 22, metastin, or metastin(45−
54) was dissolved in 50% DMSO and administered subcutaneously in
five Crl:CD(SD) male rats. Testosterone AUC0−4h was measured
using blood samples (0.4 mL) obtained from the tail vein of 22-,
metastin-, or metastin(45−54)-treated animals before administration
(0 h) and 0.5, 1, 2, 4, and 8 h after administration. Each bar represents
the mean standard deviation (n = 6). A pound sign and two asterisks
indicate p ≤ 0.025 (one-tailed Shirley−Williams test) and p ≤ 0.01
(Student’s t test) vs vehicle (0 nmol/kg) group, respectively.
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DCM (1/5/19/75) was added. The mixture was shaken for 10 min,
and the solution was distilled off. This procedure was repeated until
yellow coloration, caused by the free Mtt group in the TFA/TIS/TFE/
DCM (1/5/19/75) mixture, disappeared when the solution was
added, indicating that the Mtt group had been removed.
testosterone levels and led us to select 22 as an investigational
agent for the treatment of sex-hormone-dependent diseases.
EXPERIMENTAL SECTION
■
The resulting Fmoc-Orn-Trp(Boc)-Rink amide MBHA resin was
neutralized with 5% DIEA/DCM solution. After washing with DCM,
25 mL of DCM/TFE (4:1) and 1.946 g (6 mmol) of N-methyl-N,N′-
bis-Boc-1-guanylpyrazole were added to it. DIEA was added to the
mixture to adjust the pH of the solution to 10, and the mixture was
shaken for 15 h to give Fmoc-Arg(Boc2,Me)-Trp(Boc)-Rink amide
MBHA resin. Fmoc-Leu was introduced into the resin as described
above. The Fmoc group was removed from the subsequent Fmoc-Leu-
Arg(Boc2,Me)-Trp(Boc)-Rink amide MBHA resin (2 mmol) to give
H-Leu-Arg(Boc2,Me)-Trp(Boc)-Rink amide MBHA resin (2 mmol).
Separately, 2.326 g (8 mmol) of Fmoc-NHNH2·HCl was suspended
in 20 mL of DMF. Under ice cooling, a suspension of 297 mg (8
mmol) of CDI in 20 mL of THF and 2.787 mL (16 mmol) of DIEA
was added to the first suspension, followed by stirring at room
temperature for 1 h. The resulting reaction solution was added to the
H-Leu-Arg(Boc2,Me)-Trp(Boc)-Rink amide MBHA resin described
above. The mixture was stirred for 15 h at room temperature. The
resultant resin was washed and dried to give 6.394 g of Fmoc-azaGly-
Leu-Arg(Boc2,Me)-Trp(Boc)-Rink amide MBHA resin.
After 3.197 g (1 mmol) of the resin obtained was swollen in DMF,
the resin was treated with 30 mL of 20% piperidine/DMF solution for
20 min to remove the Fmoc group. The resin obtained was washed
with DMF and treated with 1.806 g (4 mmol) of Trt-Phe-OH·
0.5AcOEt, 2.086 g (4 mmol) of PyAOP, 8 mL (4 mmol) of 0.5 M
HOAt/DMF, and 2.787 mL (16 mmol) of DIEA for 90 min at room
temperature to give Trt-Phe-azaGly-Leu-Arg(Boc2,Me)-Trp(Boc)-
Rink amide MBHA resin. The resin obtained was washed with
DCM, and after swelling, 30 mL of TFA/TIS/TFE/DCM (1/5/19/
75) was added. The mixture was shaken for 10 min, and the solution
was distilled off. This procedure was repeated until yellow coloration
caused by the free Trt group in the TFA/TIS/TFE/DCM (1/5/19/
75) mixture disappeared when the solution was added, indicating that
the Trt group had been removed. The H-Phe-azaGly-Leu-Arg-
(Boc2,Me)-Trp(Boc)-Rink amide MBHA resin obtained was neutral-
ized with 5% DIEA/DMF solution and washed with DMF. Thereafter,
the resin was treated with 1.590 g (4 mmol) of Fmoc-Thr(t-Bu)-OH,
0.636 mL (4 mmol) of DIPCDI, and 8 mL (4 mmol) of 0.5 M HOAt/
DMF for 90 min at room temperature to introduce Thr(t-Bu).
Subsequently, Fmoc deprotection was performed by treatment with 30
mL of 20% piperidine/DMF solution for 20 min and condensation by
the DIPCDI/HOAt method, similar to the introduction of Thr(tBu);
this was repeated so that Asn(Trt), D-Trp(Boc), and D-Tyr(t-Bu) were
introduced to give Fmoc-D-Tyr(t-Bu)-D-Trp(Boc)-Asn(Trt)-Thr(t-
Bu)-Phe-azaGly-Leu-Arg(Boc2,Me)-Trp(Boc)-Rink amide MBHA
resin. The resin obtained was treated with 30 mL of 20%
piperidine/DMF solution for 20 min to remove the Fmoc group.
The resin obtained was washed to give H-D-Tyr(t-Bu)-D-Trp(Boc)-
Asn(Trt)-Thr(t-Bu)-Phe-azaGly-Leu-Arg(Boc2,Me)-Trp(Boc)-Rink
amide MBHA resin. The resin obtained was treated with 188.7 μL (2
mmol) of Ac2O and 348.4 μL (2 mmol) of DIEA in 20 mL of DMF
for 30 min at room temperature to acetylate the N-terminus. The resin
was then washed and dried to give 4.168 g of Ac-D-Tyr(t-Bu)-D-
Trp(Boc)-Asn(Trt)-Thr(t-Bu)-Phe-azaGly-Leu-Arg(Boc2,Me)-Trp-
(Boc)-Rink amide MBHA resin.
Instruments and Materials. Peptide synthesis was carried out
with an ABI 433A and/or manual shaker using Fmoc chemistry. Nα-
Fmoc-Rink amide MBHA resin was purchased from Calbiochem-
Novabiochem Japan, Ltd. (Tokyo, Japan) and Watanabe Chemical
Industries, Ltd. (Hiroshima, Japan). PAL resin was purchased from
Advanced Chemtech (Louisville, KY). Nα-Fmoc-(and side-chain)-
protected amino acids were obtained from Peptide Institute, Inc.
(Osaka, Japan), Kokusan Chemical Co., Ltd. (Tokyo, Japan),
Watanabe Chemical Industries, Ltd., and Calbiochem-Novabiochem
Japan, Ltd. Side-chain protections were as follows: Arg(Pbf), Asn(Trt),
Orn(Mtt), Ser(t-Bu), Tyr(t-Bu), Trp(Boc). Solvents and other
reagents were reagent grade and used without further purification
unless otherwise noted. DIPCDI, HOBt, and TFA were purchased
from Wako Chemical Industries, Ltd. (Osaka, Japan). Piperidine,
PyBOP, PyBrop, and TIS were purchased from Watanabe Chemical
Industries, Ltd. PyAOP was purchased from PerSeptive Biosystems
Inc. (Hamburg, Germany). The substitution of the resins was
determined by spectrophotometric analysis (Shimadzu UV-160A
UV/vis spectrophotometer) at 290 nm for the dibenzofulvene−
piperidine adduct formed upon deprotection of the amino-terminal
Fmoc group.
The crude peptides were purified to homogeneity by RP-HPLC.
HPLC conditions: YMC C18 column (20 × 150 mm), YMC C18
column (20 × 250 mm), or YMC C18 column (30 × 250 mm);
solvent gradient A (0.1% TFA in water), B (0.1% TFA in acetonitrile)
with the gradient indicated below; flow rate 8 mL/min for C18
column (20 × 150 mm, 20 × 250 mm), 20 mL/min for C18 column
(30 × 250 mm); UV detector 220/254 nm.
The purity of the products was characterized by analytical HPLC.
Reversed-phase and ion-exchange HPLC analyses were performed
with a Shimadzu gradient system using a MERCK Chromolith
FastGradient RP-18 end-capped column (2.0 × 50 mm) and
Phenomenex Luna 5 μm SCX 100 Å column (4.6 × 100 mm).
Prior to injection, a 1 mM DMSO solution of each peptide was diluted
with water to give a 100 μM solution; 10 and 20 μL of each solution
were injected into the HPLC system, respectively. For reversed-phase
HPLC analyses, all peptides were eluted with a linear gradient of 0−
50% acetonitrile in water containing 0.1% TFA at a flow rate of 0.5
mL/min, and the purity was >95% at wavelengths of 210 and 254 nm.
The peptide purity was also checked by ion-exchange HPLC at a flow
rate of 1.0 mL/min using a linear gradient of 0−0.5 M NaCl in 20 mM
sodium phosphate buffer (pH 7)/acetonitrile (75/25) at 210 and 254
nm. The molecular weights of the peptides were confirmed by
MALDI−TOF MS on a Voyager DE-PRO system (Applied
Biosystems, Foster City, CA).
General Procedure for Metastin Analogue Synthesis. All
peptides were synthesized by standard Fmoc-based solid-phase
methods. The obtained crude peptides were purified to homogeneity
with preparative HPLC. The purity of each peptide was ascertained by
analytical HPLC, and the structure assignment was performed by
MALDI-TOF MS. Structures, HPLC retention times, and MALDI-
TOF MS data of all peptides are shown in Table S1 of the Supporting
Information. The general procedure is described below as the synthesis
of Ac-[D-Tyr46,D-Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46−
54) (22).
After 5 g (0.4 mmol/g) of commercially available Rink amide
MBHA resin was swollen in DMF, the resin was treated with 50 mL of
20% piperidine/DMF solution for 20 min to remove the Fmoc group.
The resin was washed with DMF, and Trp(Boc) was introduced by
treating the resin with 4.213 g (8 mmol) of Fmoc-Trp(Boc)-OH,
1.272 mL (8 mmol) of DIPCDI, and 16 mL (8 mmol) of 0.5 M
HOAt/DMF solution at room temperature for 90 min. In a similar
manner, Orn(Mtt) was introduced to give 2 mmol of Fmoc-
Orn(Mtt)-Trp(Boc)-Rink amide MBHA resin. The resin obtained
was washed with DCM, and after swelling, 50 mL of TFA/TIS/TFE/
A mixture of half (2.111 g) of the resin obtained and 15 mL of
TFA/PhSMe/m-cresol/H2O/TIS/EDT (80/5/5/5/2.5/2.5) was
stirred for 90 min. Diethyl ether was added to the reaction solution,
the resulting precipitate was centrifuged, and the supernatant was
removed. This procedure was repeated twice for washing. The residue
was extracted with an aqueous acetic acid solution, and the extract was
filtered to remove the resin and lyophilized to give crude peptide
powders. Deprotection was performed on the remaining 2.111 g of the
resin under the same conditions to give about 650 mg of crude peptide
powders in total. Approximately 50 mg each of the crude peptide
obtained was purified by sequential application (60 min) at a flow rate
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of 15 mL/min with eluent A/eluent B = 71/29 to 61/39 (eluent A is
0.1% TFA in water, and eluent B is 0.1% TFA in acetonitrile) on
preparative HPLC using a YMC Pack R&D-ODS-5-B S-5, 120A
column (30 × 250 mm). The fractions containing the product were
collected and lyophilized to give 255.5 mg of white powders as the
purified sample; mass spectrum: MALDI-TOF (α-cyano-4-hydrox-
ycinnaminic acid, monoisotopic) C64H83N17O13 [M + H]+ 1298.82
(calcd 1298.64). Elution time on RP-HPLC: 11.92 min. Elution
conditions: Merck Chromolith FastGradient RP-18 end-capped
column (2.0 × 50 mm), linear density gradient elution with eluent
A/eluent B = 95/5 to 25/75 (14 min), using 0.1% TFA in water as
eluent A and 0.1% TFA in acetonitrile as eluent B; flow rate 0.5 mL/
min. Elution time on IE-HPLC: 7.27 min. Elution conditions:
Phenomenex Luna 5 μm SCX 100 Å column (4.6 × 100 mm), linear
density gradient elution with eluent A/eluent B = 100/0 to 0/100 (20
min) using 20 mM sodium phosphate buffer (pH 2.1)/acetonitrile
(75/25) as eluent A and 0.5 M NaCl in 20 mM sodium phosphate
buffer (pH 2.1)/acetonitrile (75/25) as eluent B; flow rate 1.0 mL/
min.
Calcium Mobilization Assay. Intracellular calcium flux was
measured as the increase in fluorescence emitted by the calcium-
binding fluorophore, fluo-3. Human KISS1R-transfected CHO cells
(hKISS1R) were seeded into 96-well plates (Corning International,
Tokyo, Japan) at 15 000 cells/mL and cultured for 24 h until they
were used in the functional FLIPR assays. The wells were then loaded
with 100 μL of HANKS/HBSS−probenecid−FBS solution containing
fluo-3 (0.5 μg, Molecular Probes), DMSO (2.1 μL), and pluronic acid
(2.1 μL) for 1 h at 37 °C (5% CO2).
HANKS/HBSS−probenecid−FBS solution was prepared by mixing
HANKS/HBSS (21 mL, prepared by mixing HANKS (9.8 g) and
sodium bicarbonate (0.35 g) with 1 M HEPES (20 mL) and adjusting
the solution to pH 7.4 with 1 N NaOH, followed by sterilization) with
250 mM probenecid (210 μL) and FBS (210 μL). The cells were
washed four times with washing buffer (HANKS/HBSS containing 2.5
mM probenecid). After the final wash, 100 μL of residual volume
remained in each well of the cell plate. Peptides were dissolved in
DMSO to form 1 mM stock solutions and diluted in buffer (HANKS/
HBSS containing 2.5 mM probenecid, 0.2% BSA, and 0.1% CHAPS)
in 96-well sample plates (V-Bottom plate type 3363, Corning
International, Tokyo, Japan). The FLIPR (Molecular Devices) was
programmed to transfer 50 μL from each well of the sample
microplate to each well of the cell plate and to record fluorescence for
3 min in 1 s intervals during the first minute and 3 s intervals during
the last 2 min. Peak fluorescence counts from the 0 to 180 s time
points were used to determine agonist activity. The instrument
software normalized the fluorescent reading to give equivalent initial
readings at time zero.
Pharmacokinetics of Metastin Analogues in Rats. The
pharmacokinetics of the metastin analogues was evaluated using 10
groups of Crl:CD(SD) rats (3 rats/group; Charles River Laboratories
Japan, Inc.). Compound 4, 14, 22, 26, or 27 was given intravenously
or subcutaneously to rats at a dose of 1 mg/kg. Blood samples (100
μL) were collected 5, 10, 15, and 30 min, and 1, 2, 4, 8, and 24 h after
injection to determine the concentration of each compound. The
plasma was obtained by removing plasma proteins with 0.2% formic
acid/methanol (1:4, v/v) and acetonitrile, followed by centrifugation
at 15 000 rpm for 5 min. The obtained supernatant was treated with
0.4% aqueous formic acid and centrifuged at 15 000 rpm for 5 min.
The plasma concentration of compound 4, 14, 22, 26, or 27 was
determined by LC−MS. The pharmacokinetic parameters associated
with each group were assessed using noncompartmental analysis. The
C5min, AUC0−24h, MRT, Vdss, and CLtotal were obtained for each rat
after intravenous administration. Cmax, Tmax, AUC0−24h, MRT, and BA
were also determined for each rat after subcutaneous administration.
Chemical Stability of Metastin Analogues in Aqueous
Solutions. Aqueous solutions of compound 1, 13, 14, 22, 26, or 27
at the desired pH, pH 1.2 (JP1, first fluid for the disintegration test;31
containing 10% acetonitrile), 3, 5, or 7, were stored in the dark at 37
°C. At preselected times the reaction mixtures were analyzed by
HPLC. The chromatograms were achieved using CAPCELLPAK C18
MG (250 × 4.6 mm) eluted with a 10−50% linear gradient of solvent
B over 20 min at a flow rate of 1.0 mL/min. Solvent A comprised
H2O/0.05 mol/L AcONH4(aq)/acetonitrile (8:1:1), and solvent B
was 0.05 mol/L AcONH4(aq)/acetonitrile (1:9). Peptides were
detected spectrophotometrically at 230−280 nm. The peptide
mixtures were incubated at 37 °C for up to 2 weeks, depending on
the experiment. The quantity of peptide in the buffer solutions was
determined from the area under the HPLC peaks. The percentage of a
given compound 1, 13, 14, 22, 26, or 27 recovered from the buffer
solutions after incubation (stability index) was calculated relative to
the amount of peptide at time 0 (100%).
Evaluation of the Effect of Metastin Peptide Derivatives on
Blood Testosterone Levels in Mature Male Rats. A metastin
peptide derivative (hereinafter peptide) was dissolved in distilled water
(Otsuka Pharmaceutical Factory, Inc.) or 50% DMSO to prepare 2, 1,
0.5, 0.3, 0.1, 0.05, 0.03, or 0.01 mM peptide solutions and loaded into
5 ALZET osmotic pumps (0.2 mL volume, release rate 0.001 mL/h,
model 2001, DURECT Corp., Cupertino, CA). The filled ALZET
pumps were implanted subcutaneously into the back of 9-week old
male Crl:CD(SD) rats (n = 5; Charles River Laboratories Japan, Inc.)
under ether anesthesia, one pump per animal. For negative controls,
distilled water (Otsuka Pharmaceutical Factory, Inc.) was used in five
ALZET osmotic pumps, which were similarly implanted into five rats.
These pump-implanted rats were fed for 6 days under normal feeding
conditions. After weighing, the animals were decapitated, and blood
samples were collected. After 0.03 mL of aprotinin solution (Trasylol,
Bayer) containing 0.1 mg/mL EDTA·2Na was added to each milliliter
of blood, the mixture was centrifuged at 1800g for 25 min to recover
the plasma. From the obtained plasma, 0.05 mL was applied to RIA
(Siemens Healthcare Diagnostics Inc., Deerfield, IL) to measure the
plasma testosterone level of each rat. Values less than the lower limit of
detection (0.04 ng/mL) in RIA were treated as 0.04. Mean values of
the testosterone levels from five rats receiving peptide treatment were
calculated as a percentage of the control values.
Single Administration of 22, a Metastin Agonist, Induced
Marked LH and Testosterone Release in Male Rats. Animals.
This study was performed on two consecutive days. In each
experiment, nine male Crl:CD(SD) rats (Charles River Laboratories
Japan, Inc.) were randomly divided into seven groups (Group 1: 22, 1
nmol/kg; Group 2: 22, 0.1 nmol/kg; Group 3: 22, 0.01 nmol/kg;
Group 4: metastin, 10 nmol/kg; Group 5: metastin, 1 nmol/kg; Group
6: metastin(45−54), 1000 nmol/kg; group 7, vehicle control; n = 3)
based on body weight 1 day before administration. Drug solutions
were administered subcutaneously, and blood samples (0.4 mL) were
obtained via the tail vein before administration (0 h) and 0.5, 1, 2, 4,
and 8 h after administration. Plasma samples were prepared by
collecting the blood samples in plastic tubes containing 4 IU of sodium
heparin (Ajinomoto Pharmaceuticals Co., Ltd. Tokyo, Japan) and
centrifugation. Data obtained from two independent experiments were
combined. Animal experiments were approved by the Takeda
Experimental Animal Care and Use Committee, in accordance with
NIH standards.
Chemicals. Metastin(45−54) (code no. 4389-v, lot no. 560213)
was purchased from Peptide Institute, Inc., Kyoto, Japan. The vehicle
was 1:4 (v/v) 20% DMSO (Sigma-Aldrich, St. Louis, MO)/saline
(Otsuka Pharmaceutical Factory, Inc.). Compound 22, metastin, or
metastin(45−54) solutions were prepared using this vehicle.
Endocrine Measurements. Plasma LH and total testosterone were
determined using a commercially available RIA kit by T.N. Technos,
Ltd. (LH: Rat LH [125I] RIA System, GE Healthcare; total
testosterone: DPC Total Testosterone Kit, Diagnostic Products
Corp.). The value of plasma LH below the detection limit (0.78 ng/
mL) was presumed to be 0.78 ng/mL. The detection limit of plasma
testosterone was 0.04 ng/mL.
Statistics. After baseline (0 h) LH or testosterone levels were
subtracted, the effect of LH or testosterone release was expressed as
the area under the curve between 0 and 4 h, i.e., LH AUC0−4h or
testosterone AUC0−4h, after administration. LH AUC0−4h was analyzed
by the one-tailed Shirley−Williams test (22- or metastin-treated group
vs vehicle-treated group) or Aspin−Welch t test (metastin[45−54]-
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treated group vs vehicle-treated group). Testosterone AUC0−4h was
analyzed by one-tailed Williams’s test (22- or metastin-treated group
vs vehicle-treated group) or Student’s t test (metastin[45−54]-treated
group vs vehicle-treated group). The differences were considered
significant for p ≤ 0.05 (Student’s and Aspin−Welch t test) or p ≤
0.025 (Williams’s test or Shirley−Williams test).
M. Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-
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Vanderwinden, J. M.; De Poul, E.; Brezillon, S.; Tyldesley, R.; Suarez-
Huerta, N.; Vandeput, F.; Blanpain, C.; Schiffmann, S. N.; Vassart, G.;
Parmentier, M. The metastasis suppressor gene KiSS-1 encodes
kisspeptins, the natural ligands of the orphan G protein-coupled
receptor GPR54. J. Biol. Chem. 2001, 276, 34631−34636.
(3) Muir, A. I.; Chamberlain, L.; Elshourbagy, N. A.; Michalovich, D.;
Moore, D. J.; Calamari, A.; Szekeres, P. G.; Sarau, H. M.; Chambers, J.
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Emson, P.; Faull, R.; Philpott, K. L.; Harrison, D. C. AXOR12, a novel
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ASSOCIATED CONTENT
* Supporting Information
Table S1 lists the chemical data for decapeptide and
nonapeptide metastin analogues. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: taiji.asami@takeda.com. Phone: +81-466-32-1186.
Fax: +81-466-29-4453.
■
(4) Roa, J.; Aguilar, E.; Dieguez, C.; Pinilla, L.; Tena-Sempere, M.
New frontiers in kisspeptin/GPR54 physiology as fundamental
gatekeepers of reproductive function. Front. Neuroendocrinol. 2008,
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and GPR54 in the neuroendocrine regulation of reproduction. Annu.
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(6) Roseweir, A. K.; Millar, R. P. The role of kisspeptin in the control
of gonadotrophin secretion. Hum. Reprod. 2009, Update 15, 203−212.
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Peripheral administration of metastin induces marked gonadotropin
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge Stephen Mosley and Tamara Bailey of
FireKite for editing assistance in the development of this
manuscript. This study was funded by Millennium: The Takeda
Oncology Co.
ABBREVIATIONS USED32
■
[Ca2+]i, calcium ion concentration; Abu, 2-aminobutyric acid;
AcOEt, ethyl acetate; Arg(Boc2,Me), Nω,ω′-bis(tert-butoxycar-
bonyl)-Nω-methylarginine; Arg(Me), Nω-methylarginine; Asu,
aminosuccinimidyl; azaGly, azaglycine; BA, bioavailability; CDI,
1,1′-carbonyldiimidazole; CHAPS, 3-[(3-cholamidopropyl)-
dimethylammonio]-1-propanesulfonate; CHO, Chinese ham-
ster ovary; C5min, plasma concentration at 5 min after
administration; CLtotal, total body clearance; Cmax, maximum
plasma concentration; DIEA, N,N-diisopropylethylamine;
DIPCDI, diisopropylcarbodiimide; EDT, 1,2-ethanedithiol;
FBS, fetal bovine serum; FLIPR, fluorometric imaging plate
reader; HANKS/HBSS, Hank’s balanced salt solution; HCl,
hydrochloric acid; HEPES, 4-(2-hydroxyethyl)-1-piperazinee-
thanesulfonic acid; hKISS1R, human KISS1R-expressing CHO
cells; HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, N-hydrox-
ybenzotriazole; HPG, hypothalamic−pituitary−gonadal; Hyp,
hydroxyproline; KISS1R, KISS1 receptor; MBHA resin, 4-
methylbenzhydrylamine hydrochloride salt resin; MRT, mean
residence time; NaOH, sodium hydroxide; Orn, ornithine; PAL
resin, peptide amide linker resin; Pbf, 2,2,4,6,7-pentamethyldi-
hydrobenzofuran-5-sulfonyl; Phg, phenylglycine; PhSMe, thio-
anisole; Pya(4), 4-pyridylalanine; PyAOP, ((7-azabenzotriazol-
1-yl)oxy)tripyrrolidinophosphonium hexafuorophosphate;
PyBOP, (1H-benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate; PyBrop, bromotripyrrolidinophospho-
nium hexafluorophosphate; RP-HPLC, reversed-phase high-
performance liquid chromatography; TFE, 2,2,2-trifluoroetha-
nol; TIS, triisopropylsilane; Tle, tert-leucine; Tmax, time to
maximum plasma concentration; Trt, triphenylmethyl (trityl);
(15) Asami, T.; Nishizawa, N.; Kanehashi, K.; Ishibashi, Y.;
Horikoshi, Y.; Nakayama, M.; Tarui, N.; Matsumoto, H.; Ohtaki, T.;
Kitada, C. Trypsin resistance of a decapeptide KISS1R agonist with
Nω-methylarginine. Bioorg. Med. Chem. Lett. 2012, 20, 6328−6332.
(16) Asami, T.; Nishizawa, N.; Kanehashi, K.; Ishibashi, Y.;
Horikoshi, Y.; Nakayama, M.; Tarui, N.; Matsumoto, H.; Ohtaki, T.;
Kitada, C. Serum stability of selected decapeptide agonists of KISS1R
using pseudopeptides. Bioorg. Med. Chem. Lett. 2012, 20, 6391−6396.
VdSS, volume of distribution at steady state
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